De novo-designed translation-repressing riboregulators for multi-input cellular logic

Abstract

Efforts to construct synthetic biological circuits with more complex functions have often been hindered by the idiosyncratic behavior, limited dynamic range and crosstalk of commonly utilized parts. Here, we employ de novo RNA design to develop two high-performance translational repressors with sensing and logic capabilities. These synthetic riboregulators, termed toehold repressors and three-way junction (3WJ) repressors, detect transcripts with nearly arbitrary sequences, repress gene expression by up to 300-fold and yield orthogonal sets of up to 15 devices. Automated forward engineering is used to improve toehold repressor dynamic range and SHAPE-Seq is applied to confirm the designed switching mechanism of 3WJ repressors in living cells. We integrate the modular repressors into biological circuits that execute universal NAND and NOR logic and evaluate the four-input expression NOT ((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These capabilities make toehold and 3WJ repressors valuable new tools for biotechnological applications.

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Fig. 1: Operating mechanisms of de novo-designed repressors and in vivo characterization.
Fig. 2: Characterization of forward-engineered toehold repressors.
Fig. 3: In-cell SHAPE-Seq confirmation of the 3WJ repressor mechanism.
Fig. 4: Assessment of toehold and 3WJ repressor orthogonality.
Fig. 5: Two-input logic operations using repressor-based devices.
Fig. 6: Multi-input ribocomputing devices employing toehold and 3WJ repressors.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. Supplementary Tables are available from A.A.G. in spreadsheet format upon request. The following plasmids from the study are available from Addgene: pYZ_3WJrep_N20_switch 132722; pYZ_3WJrep_N19_switch 132723; pYZ_3WJrep_N10_switch 132724; pYZ_3WJrep_N24_switch 132725; pYZ_3WJrep_N20_trigger 132726; pYZ_3WJrep_N19_trigger 132727; pYZ_3WJrep_N10_trigger 132728; pYZ_3WJrep_N24_trigger 132729; pYZ_NAND2_L17_S19_S11 132730; pYZ_NAND3_L11_S11_S13_S19 132731; pYZ_NAND4_L17_S24_S11_S19_S13 132732; pYZ_3WJrep_N11_trigger 132733; pYZ_3WJrep_N12_trigger 132734; pYZ_3WJrep_N13_trigger 132735; pAG_PluxB_ToeRep_N01_trigger 132736; pAG_Ptet*_ToeRep_N01_switch 132737; pYZ_PluxB_3WJrep_N19_trigger 132738; pYZ_Ptet*_3WJrep_N19_switch 132739; pAG_ToeRep_N09_trigger 132740; pAG_ToeRep_N09_switch 132741; pJK_ToeRepG2_N02_switch 132742; pJK_ToeRepG2_N64_switch 132743; pJK_ToeRepG2_N19_switch 132744; pJK_ToeRepG2_N02_trigger 132745; pJK_ToeRepG2_N64_trigger 132746; pJK_ToeRepG2_N19_trigger 132747.

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Acknowledgements

This work was supported by an NIH Director’s New Innovator Award (1DP2GM126892), an Alfred P. Sloan Research Fellowship (FG-2017-9108), Gates Foundation funds (OPP1160667), an Arizona Biomedical Research Commission New Investigator Award (ADHS16-162400), a DARPA Young Faculty Award (D17AP00026), Gordon and Betty Moore Foundation funds (#6984), NIH funds (1R21AI136571) and Arizona State University funds to A.A.G.; an NIH Director’s Pioneer Award (1DP1GM133052-01), Office of Naval Research funds (N00014-16-1-2410) and NSF funds (CCF-1317291, MCB-1540214) to P.Y.; an NSF CAREER award to J.B.L. (1452441); Defense Threat Reduction Agency funds (HDTRA1-14-1-0006), Air Force Office of Scientific Research funds (FA9550-14-1-0060) and Paul G. Allen Frontiers Group funds to J.J.C.; and BMBF funds (Erasynbio project UNACS -031L0011) and DFG funds (SFB 10327TPA2) to F.C.S. J.K. acknowledges a Wyss Institute Director’s Cross-Platform Fellowship. The views, opinions and/or findings contained in this article are those of the authors and should not be interpreted as representing the official views or policies, either expressed or implied, of the NIH, DARPA or the Department of Defense.

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J.K. and A.A.G. designed the toehold repressors and ribocomputing devices. J.K., M.T. and A.A.G. performed experiments for the toehold repressors and their ribocomputing devices. Y.Z., S.C. and A.A.G. designed the 3WJ repressors and ribocomputing devices. Y.Z. and S.C. performed experiments for the 3WJ repressors and their ribocomputing devices. P.D.C. performed SHAPE-Seq measurements. J.K., Y.Z., A.A.G. and P.D.C. analysed the data. J.K., Y.Z., P.D.C. and A.A.G. wrote the manuscript. J.K., A.A.G., P.D.C., J.B.L. and P.Y. edited the manuscript. A.A.G., P.Y., J.B.L., J.J.C., P.A.S. and F.C.S. supervised the research.

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Correspondence to Peng Yin or Alexander A. Green.

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US provisional patents have been filed by J.K., A.A.G., J.J.C. and P.Y. and by Y.Z. and A.A.G., based on this work. P.Y. is the co-founder of Ultivue Inc. and NuProbe Global.

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Kim, J., Zhou, Y., Carlson, P.D. et al. De novo-designed translation-repressing riboregulators for multi-input cellular logic. Nat Chem Biol 15, 1173–1182 (2019). https://doi.org/10.1038/s41589-019-0388-1

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